The role of small proteins in metabolic regulation of cyanobacteria

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The cyanobacterium Synechocystis sp. strain PCC 6803 serves as the model for oxygenic photosynthesis as well as metabolic regulation within this environmentally important group of primary producers. Recently, previously unknown transcripts were identified in the Synechocystis genome that could encode small proteins. Within SPP2002, we aim to analyze whether or not the newly discovered small proteins are involved in metabolic regulation of cyanobacteria. Three small proteins namely Norf4 (31 AA), pSYS_ORF3/AcnSP (44 AA) and HliR1 (37 AA) were chosen for functional annotation. Biochemical and physiological analysis using mutants (knock out and selected site-specific mutants abolishing the small protein expression) and strains ectopically expressing the small proteins will reveal if they are really functionally important. We hypothesize that these small proteins could directly or indirectly regulate specific enzymes such as glyceraldehyde 3-phosphate dehydrogenase (Gap) and aconitate dehydratase (AcnB) or participate in the regulation of stress protection of cyanobacteria. During the first funding period we demonstrated that pSYS_ORF3 regulates the biochemical activity of the tricarboxylic acid enzyme AcnB, therefore it was renamed as AcnSP. Our results indicated that the small protein AcnSP impacts the carbon flow into the oxidative branch of the open cyanobacterial TCA cycle thereby regulating the overall C/N metabolism in Synechocystis. Furthermore, our results demonstrated that sORFs can originate even from seemingly irregular gene fragment duplications and add another element to the complex regulatory system around AcnB. Moreover, we obtained clear hints that AcnSP is also involved in the regulation of iron-stress response, which is consistent with the dual role of AcnB as TCA cycle enzyme and iron-sensory module in many other organisms. Unravelling the underlying mechanisms, i.e. how AcnSP is involved in the acclimation to iron stress probably participating in a network with sRNAs and RNase E action on acnB mRNA stability, will be in the focus of the upcoming project period.Our work on Norf4 and HliR1 verified that these small proteins are indeed involved in metabolic regulation or stress acclimation of Synechocystis. The norf4 mutant is unable to grow with glucose in darkness and shows alterations in the utilization of stored glycogen. These findings are consistent with our initial hypothesis that Norf4 is regulating the activity of the glycolytic key enzyme Gap1. Finally, the hliR1 mutant showed a clear high-light-sensitive phenotype due to reduced superoxide dismutase B (SodB) activity. This finding is supporting our initial hypothesis that HliR1 is regulating SodB, which plays a crucial role in the acclimation of cyanobacteria towards oxidative stress induced by high-light or disturbed iron homeostasis. The molecular mechanisms of the Norf4- and HliR1-mediated regulations will be analyzed in the future.